The transcription factor ATF3 switches cell death from apoptosis to necroptosis in hepatic steatosis in male mice

Hepatocellular death increases with hepatic steatosis aggravation, although its regulation remains unclear. Here we show that hepatic steatosis aggravation shifts the hepatocellular death mode from apoptosis to necroptosis, causing increased hepatocellular death. Our results reveal that the transcription factor ATF3 acts as a master regulator in this shift by inducing expression of RIPK3, a regulator of necroptosis. In severe hepatic steatosis, after partial hepatectomy, hepatic ATF3-deficient or -overexpressing mice display decreased or increased RIPK3 expression and necroptosis, respectively. In cultured hepatocytes, ATF3 changes TNFα-dependent cell death mode from apoptosis to necroptosis, as revealed by live-cell imaging. In non-alcoholic steatohepatitis (NASH) mice, hepatic ATF3 deficiency suppresses RIPK3 expression and hepatocellular death. In human NASH, hepatocellular damage is correlated with the frequency of hepatocytes expressing ATF3 or RIPK3, which overlap frequently. ATF3-dependent RIPK3 induction, causing a modal shift of hepatocellular death, can be a therapeutic target for steatosis-induced liver damage, including NASH.

7. In the overexpression models, a single adenoviral infection was performed. Did the expression of ATF3 persist? Was the expression preferentially in hepatocytes, or was it ubiquitous throughout the liver? 8. Recent manuscripts show that ATF3 promotes NASH (Tu et al, Hepatology 2020)-the authors need to reconcile the literature with their findings that ATF3 loss did not seem to impact steatosis development in Extended Data Figure 2. 9. The results section would greatly benefit from additional introduction of the methods/models used, particularly in vivo, in order to improve the ease of understanding and clarity for readers.
Reviewer #2 (Remarks to the Author): The manuscript from Inaba et al. titled 'ATF3 switches cell death from apoptosis to necroptosis in hepatic steatosis' demonstrated that increased hepatic steatosis severity shifts the hepatocellular death mode from apoptosis to necroptosis, aggravating the liver damage. In vivo, ATF3 regulates the expression of RIPK3. Under severe hepatic steatosis conditions, hepatic ATF3-deficient oroverexpressing mice display decreased or increased necroptosis, respectively. In vitro studies implicate that ATF3 increases RIPK3 expression by directly binding to its gene promoter leading to necroptosis. In mice and patients with NASH disease severity appears to associate with ATF3 and RIPK3 expression in hepatocytes. This study points out a novel function of ATF3 related to necroptosis in liver steatosis injury.
However, some issues remain to be considered: 1. The authors indicated that ATF3 is induced in hepatocytes during liver stetaosis injury. What is exactly the mechanism associated with this increased expression?

REVIEWER COMMENTS
Reviewer #1 (Remarks to the Author): In the manuscript 'ATF3 switches cell death from apoptosis to necroptosis in hepatic steatosis' the authors analyze the mechanisms that drive necroptosis during the progression of NASH.
They initially show that mice with severe steatosis have increased necroptosis following hepatectomy, and this could be limited by knockdown of RIPK3, a key driver of necroptosis.
The key finding of this paper is the novel regulation of necroptosis through ATF3-mediated upregulation of RIPK3, shown through mouse models and in vitro data using primary hepatocytes and an interesting model of hepatoma cells where the Ripk3 gene requires demethylation for ATF3 to promote its expression. Increased ATF3 expression during the progression of steatosis is suggested to be a switch that drives necroptosis as opposed to apoptosis.
While different mouse models are utilized, and the in vitro data is thorough, several issues limit the clarity and impact of this work. The use of a hepatectomy model without clear justification distracted from the major conclusions regarding the increased necroptosis in hepatic steatosis.
We apologise for the lack of a thorough explanation and clear description of why we used a hepatectomised model of high-fat diet (HFD) feeding, in addition to a NASH model of methionine/choline-deficient diet (MCD) feeding. The aim of this study was to clarify the regulation of hepatocellular death during regeneration after acute and chronic liver damage in hepatic steatosis, as hepatocellular death during regeneration exacerbates both types of fatty liver damage. Because hepatectomy models are a 'clean' model for studying acute regeneration without any direct effects from the causes of liver damage (e.g., acetaminophen, carbon tetrachloride, ischaemia-reperfusion), as reported previously 1,2 , we used a hepatectomy model to investigate hepatocellular death during hepatic regeneration after acute damage. To the Introduction, we have added the following: 'we investigated the mode of hepatocellular death and its regulatory mechanism during liver regeneration after liver damage by use of hepatectomised hepatic steatosis mice as models of acute liver damage and by use of MCD-induced NASH mice as models of chronic liver damage.' To the Results, we have added the following: 'Specifically, in 2-week or 16-week HFD-fed obese mice, we performed 70% partial hepatectomy, which is considered a 'clean' model of acute liver regeneration 5 .' Furthermore, to the first paragraph of the Discussion, we have added the following: 'Despite the importance of the lytic cell death of hepatocytes, it remained unclear what kind of lytic cell death is selected, and how, in steatotic hepatocytes after acute or chronic liver damage. In this study, we investigated the modes of hepatocellular death in hepatectomised mice with HFD-induced hepatic steatosis as models of acute liver damage and in MCD-induced NASH mice as models of chronic liver damage. We found that the mode of hepatocellular death shifts from apoptosis to necroptosis as the hepatic steatosis is exacerbated and that the eIF2α signalling-inducing transcription factor ATF3 determines this modal shift through RIPK3 induction. We also determined that ATF3-dependent RIPK3 induction plays important roles both in acute injury of steatotic liver and NASH' Indeed, necroptosis in the severe steatosis model in Figures 1 and 2 is not analyzed. In addition, key markers of necroptosis (p-MLKL) were not analyzed in several experiments, relying only on mRNA analyses of total MLKL. The potential role of ferroptosis, which is also regulated in part by ATF3, is also not investigated. The impact of the manuscript in its current form is judged to be moderate, but to a potentially limited audience.
In response to the reviewer's suggestion, we newly measured MLKL phosphorylation In accordance with our apology above, we agree that the original manuscript lacked an explanation of why we used a hepatectomy model in addition to a NASH model of MCD feeding. Because the aim of this study was to clarify the regulation of hepatocellular death after acute and chronic liver damage in hepatic steatosis, we have added a description of this aim to the Introduction, Results and Discussion.
In terms of the rationale for using a hepatectomised HFD model and MCD-NASH model While both high-fat high-cholesterol diet (HFHC) feeding for 28 weeks and MCD feeding for 6 weeks result in NASH with hepatic inflammation and fibrosis 28 , MCD feeding evoked a sufficiently potent activation of eIF2α signalling to induce the protein and mRNA expression of ATF3 and RIPK3, as in hepatectomised HFD-induced hepatic steatosis, but not with HFHC feeding (Fig. 8a, b). RIPK3 and MLKL phosphorylation was also increased by MCD feeding (Fig. 8a).' To the Discussion, we have added the following: 'Furthermore, 28-week HFHC feeding, which is used to create a NASH model 28 , was associated with low expression of ATF3 and RIPK3. Under these conditions, the hepatocellular stress may be too weak to induce ATF3 and RIPK3.' 3. The authors analyzed MLKL in whole liver using qPCR, but did not show levels of total or phosphorylated MLKL. Indeed, p-MLKL is missing from several in vivo and in vitro analyses.
We measured the protein expression of MLKL and phospho-MLKL in all of the mouse models. Specifically, we have added the data concerning the hepatectomised HFD model to We also measured phospho-RIPK3 and MLKL in isolated hepatocytes from lean and obese mice with knockdown of ATF3 or RIPK3. However, their levels were undetectable in these hepatocytes. Therefore, to the Results, we have added the following: 'Phosphorylation of MLKL and RIPK3 was not detected in these isolated hepatocytes from both lean and obese mice (Fig. 6d).' 4. Both TNF and Caspase-8 are a key regulator of necroptosis in mice. The authors should 1) analyze whether the increased necroptosis is dependent on, or independent of, caspase-8 levels, and 2) determine whether ATF3 upregulation is downstream of TNF.
1) We measured cleaved-caspase 8 (Cl-CASP8) in vivo and in vitro. We found that Cl-CASP8 had the same tendency as cleaved-caspase 3 (Cl-CASP3) in mouse models. We have shown the in vivo data of Cl-CASP8 in the respective figures showing MLKL and phospho-MLKL, which were described in our response to comment 3.
We also measured Cl-CASP8 in H4IIE hepatoma cells the death of which is induced by and its activation by TNFα are produced independently in hepatic steatosis.' 5. The authors should assess the potential contribution of ferroptosis in their models, particularly since ATF3 can also promote ferroptosis.
We thank the reviewer for this suggestion. To investigate ferroptosis in our models, we We have examined the hepatic specificity of siRNA knockdown and found that this method of knockdown displayed moderate specificity to hepatocytes in the liver, as shown in a new Supplementary Fig. 2a- Fig. 2a-e).' Regarding the decrease in TNFα with R-KD, we believe that this decrease was due to the decrease in hepatic necroptosis. Although TNFα is a major inducer of necroptosis, lytic cell death, such as that of necroptosis, is also known to induce inflammation around dead cells by releasing pro-inflammatory intracellular molecules called damage-associated molecular patterns (DAMPs). Therefore, there is a vicious cycle of necroptosis and inflammation. R-KD can break this cycle, resulting in a decrease in hepatic TNFα expression.
To the Discussion, we have added the following: 'Given that TNFα leads to RIPK3 activation and necroptosis induction, which increases inflammation and TNFα levels As the reviewer pointed out, the original manuscript is hard to understand due to the use of multiple models. Therefore, we have revised the Results section, particularly the subheadings of each paragraph, to improve reader understanding of the model addressed in each paragraph. We have also added a further explanation and conclusion to each paragraph.
Reviewer #2 (Remarks to the Author): The manuscript from Inaba et al. titled 'ATF3 switches cell death from apoptosis to necroptosis in hepatic steatosis' demonstrated that increased hepatic steatosis severity shifts the hepatocellular death mode from apoptosis to necroptosis, aggravating the liver damage.
In vivo, ATF3 regulates the expression of RIPK3. Under severe hepatic steatosis conditions, hepatic ATF3-deficient or -overexpressing mice display decreased or increased necroptosis, respectively. In vitro studies implicate that ATF3 increases RIPK3 expression by directly binding to its gene promoter leading to necroptosis. In mice and patients with NASH disease severity appears to associate with ATF3 and RIPK3 expression in hepatocytes. This study points out a novel function of ATF3 related to necroptosis in liver steatosis injury.
However, some issues remain to be considered: 1. The authors indicated that ATF3 is induced in hepatocytes during liver steatosis injury.
What is exactly the mechanism associated with this increased expression?
The eIF2α and JNK-c-Jun signalling pathways induce ATF3 expression 3,4 To investigate 2. What is the potential involvement of specific pathways governing tissue inflammation on the role of ATF3 in liver steatosis injury? Does RNA expression data suggest that inflammatory responses are one of the key pathways involved?
In response to the reviewer's suggestion, we have examined TNFα neutralisation in severe hepatic steatosis after hepatectomy and determined that ATF3 induces RIPK3 expression and that TNFα activates this RIPK3 induction. We have added the TNFα neutralisation data to a new Fig. 5e-h and a new Supplementary Fig. 5m-o. To the Results, we have added a new paragraph entitled, 'TNFα neutralisation prevents RIPK3 phosphorylation after hepatectomy'. To the Discussion, we have added the following: 'While ATF3 is the inducer of RIPK3, TNFα is a vital activator of RIPK3 in hepatic steatosis. Indeed, neutralisation of TNFα diminished the phosphorylation of RIPK3 but did not affect ATF3 expression. Furthermore, in isolated hepatocytes, ATF3 expression induced RIPK3 expression, but did not always induce phosphorylation of RIPK3 and MLKL. These results indicate that RIPK3 induction by ATF3 and its activation by TNFα are produced independently in hepatic steatosis.' and 'Given that TNFα leads to RIPK3 activation and necroptosis induction, which increases inflammation and TNFα levels 4,9 , ATF3-dependent induction of RIPK3 in severe hepatic steatosis can cause a vicious cycle of necroptosis and inflammation.
Indeed, hepatocellular deficiency of RIPK3 or ATF3 decreased hepatic TNFα expression and ATF3 overexpression increased its expression. This vicious cycle can play an important role in the pathogenesis of the exacerbation of liver damage.' 3. In human studies, what are the mRNA levels of ATF3 and RIPK3? The human study will need to be further strengthened by additional functional analysis between ATF3/RIPK3 levels and liver injury outcomes (e.g. ALT, AST). Moreover, what are the levels of RIPK3 phosphorylation in low and high-NAS score patients?
We agree that the mRNA levels of ATF3 and RIPK3 would help to elucidate the relationship between ATF3 and RIPK3. However, we unfortunately do not have mRNA from NASH patients.
We have performed additional functional analyses comparing ATF3/RIPK3 levels and plasma aminotransferase levels. Only phospho-RIPK3 was correlated with plasma AST levels, as shown in a new Fig. 10d and Supplementary Fig. 10b. Given that ATF3 and RIPK3 enable hepatocytes to acquire a new ability to undergo necroptosis and TNFα triggers necroptosis via phospho-RIPK3, this correlation seems to be reasonable.
We also performed immunostaining of phospho-RIPK3. Phospho-RIPK3 levels were correlated with RIPK3 and ATF3 levels (Fig. 10a), plasma aminotransferase levels (Fig. 10d) and fibrosis stage (Fig. 10i), but not with NAS, steatosis, ballooning and inflammation ( Fig.   10e-h). To the paragraph named 'Hepatic ATF3 and RIPK3 expression in NASH patients', we have added an explanation of these data. 4. In the analysis of the gene expression of necroptosis-related molecules, expression of the Mlkl and Ripk1 genes was not significantly increased during the regeneration process of severely steatotic livers. Would this contradict the incresead levels of necroptosis, which is triggered by the sequential phosphorylation of RIPK3 and MLKL that disrupts the cell membrane? What additional mechanism governs RIPK3-driven necroptosis?
Thanks to the reviewers' suggestions, we have now more clearly elucidated the mechanism of the ATF3-dependent induction of necroptosis in this revised version of the manuscript. Indeed, we measured the protein expression of MLKL and phospho-MLKL in all of the mouse models. Specifically, we have added the data concerning the hepatectomised HFD model to Fig. 1f, the hepatectomised HFD model with R-KD to Fig. 2f, the hepatectomised HFD model with A-KD to Fig. 3i, the un-hepatectomised HFD model with We also performed TNFα neutralisation in a hepatectomised HFD model, as described above in our response to comment 2. Through these analyses, we found that it is TNFα that phosphorylates hepatocellular RIPK3 and MLKL.
Because MLKL is expressed in hepatocytes under basal conditions, but not RIPK3, as previously reported 5,6 , the presence of RIPK3 determines whether hepatocytes die via necroptosis or not. Indeed, in Fig. 2, we showed that R-KD decreased non-apoptotic hepatocellular death after hepatectomy in severe hepatic steatosis, indicating that TNFα should induce necroptosis in the presence of RIPK3, but apoptosis in the absence of RIPK3. Therefore, in conclusion, RIPK3 and its inducer ATF3 enable hepatocytes acquire a new ability to undergo necroptosis and TNFα triggers necroptosis via phosphorylation of RIPK3.